Phytoforensic Analysis: Thinking Outside The Box

Nearly half of all US FDA class I drug recalls since 2014 have been tainted dietary supplements adulterated with pharmaceutical ingredients.¹ Bethany Degg of The Column spoke to James Neal-Kababick, who is the Founder and Director of Flora Research Laboratories (FRL) in Oregon, USA, about phytoforensic science and his work on analyzing dietary supplements previously recalled by the US FDA.

Q. What are your main research interests?

A: Furthering phytoforensic science through educational outreach and training; and developing and implementing novel approaches to the detection of adulteration in dietary supplements. I am a big fan of microscopy, high performance thin layer chromatography (HPTLC), Fourier transform infrared (FTIR), and mass spectrometry (MS) as well as a host of other disciplines. I love bringing techniques together to help get the big picture.

Photo Credit: malerapaso/Getty Images

Q. Have dietary supplements always been regulated by the FDA? If so, how have regulations changed over the years?

A: Yes, despite the comments by many media outlets that dietary supplements are not regulated by FDA, they have always been regulated. Up until the new Dietary Supplement cGMPs (current Good Manufacturing Practices) 21 C.F.R. part 111 went into effect, the products were regulated under food GMPs. The new part 111 GMPs are much more demanding and closer to pharma GMP regulations than food regulations. In some ways, they are even more demanding because of the highly variable nature of ingredients and products.

Q. Can you describe what phytoforensic science is and why it is important?

A: Phytoforensic science is a specialized forensic science focus area that involves the application of multiple orthogonal advanced techniques from microscopy to mass spectrometry in the protection of the global food supply chain with an emphasis on dietary supplements. A driving tenant of phytoforensic science is "thinking outside the box" because some of the adulteration schemes, such as using polyphosphates to adulterate chondroitin (see our recent publication in Journal of AOAC International)² or melamine to adulterate wheat gluten, are outside of the areas that were being focused on by routine QC testing and monographing bodies.

If you consider these examples, who would think to adulterate chondroitin sulphate with Calgon detergent? Obviously, someone who figured out it would spoof the CPC titration method and boost results. More importantly, it is also something that is not detected unless you run a very specific method called Cellulose Acetate Membrane Electrophoresis (CAME), which, despite being a requirement of the USP monograph for chondroitin sulphate, has a history of not being utilized. I often teach students to put on the "bad guy" hat and think of how they would make more money if they did not care about quality, regulatory compliance, or safety: Find that trick then go look for it in your samples. I think my decades of detecting sophisticated adulteration in highly complex essential oils by gas chromatography (GC) was a great foundation for this area.

Q. Are there specific factors that manufacturers should consider when sampling products before analysis?

A: Yes, this is a very important and often poorly understood area. First, the cGMP regulations mandate that laboratory samples should be obtained using sound scientifically valid statistical sampling plans and that they represent the lot under test. That does not mean using a flour scoop to take a sample off of the top of a one ton supertote or a grab sample off a drum. There is a whole science to sampling, with detailed instructions in the United States Pharmacopeia as well as in handbooks such as Pierre Gy's "Sampling Theory".³ If your sample is not a lot representative sample, your data is meaningless as far as FDA goes.

Q. What are the chromatographic techniques used by analysts? Some argue that mass spectrometry instrumentation has advanced to the point where chromatography is no longer necessary - do you agree and why?

A: This is a great question. The heart of chromatography in our industry is high performance liquid chromatography (HPLC in all of its flavours) (ultrahigh-pressure LC [UHPLC], rapid resolution [RRLC], etc.). In addition, GC, HPTLC, and hyphenated techniques such as GC–MS and LC–MS are used. Advances in MS have been phenomenal in the last two decades I have been working with the technique. What I can do in an afternoon on our accurate mass quadrupole time-of-flight (QTOF) took weeks before. From unknown to formula to structure is faster than ever.

The direct analysis techniques such as desorption electrospray ionization (DESI), direct analysis in real time (DART), and direct sample analysis (DSA) are very exciting, and I have used DART and DSA-TOF before with great success. I think that they have a very important place in the phytoforensic laboratory and routine QC laboratory as well. Even flow injection has utility. However, until engineers design MS systems that overcome ion suppression, separation science will be a necessary and valuable partner in our routine work. In addition, the human eye and brain is an incredible pattern-matching tool. When one looks at a chromatogram and compares it to another, differences can pop out in a few seconds. This often directs the analyst right to the peak of interest. This can be done with direct analysis as well but software that helps plot ion differences and their intensity in an easy to interpret output would make that much more valuable for rapid screening. My biggest concern is missing an unknown - unknown in a sample because of ion suppression. That is why we have our MS systems connected to other detectors as well such as evaporative light scattering detector (ELSD) and diode-array detection (DAD).

Q. In a recent study you were responsible for the analysis of dietary supplements that had been previously recalled by the FDA.¹ What led you to collaborate in this investigation?

A: A significant amount of my work is focused on adulteration. Of that work, about half is focused on tainted supplements, that is, products being illegally sold as dietary supplements that contain drugs or unapproved drug analogues. The FDA defines these as unapproved drugs because legal dietary supplements are not allowed to contain drugs. Some time back, I was on the Dr. Oz Show to present data from a study we did. In that study, we found that many of the recalled tainted supplements containing drugs were still being sold in stores. I was curious to see how that has changed now that the cGMPs have been in place and enforced for a few years since then.

Q. Can you discuss the criteria used for selecting candidate dietary supplements?

A: A list was created of all the Class I recalls on products marketed as dietary supplements that contained pharmaceutical drugs or drug analogues. The exclusionary criteria included those available on Amazon, eBay, or Alibaba so we only purchased products that were sold on other websites or in stores. Of those, about 27 more had to be excluded because of label changes that did not allow us to confirm that the product in hand was the same as the one recalled (slight label changes disqualified samples). What we had left was blinded, sent to Flora Research Laboratories (our lab here in Grants Pass, Oregon, USA), and tested either with our weight loss, sports/performance, or men's virility drug screens. We did not know what product was what during testing and I only knew of the product's individual identities after the test data was sent back to Dr Cohen (lead author of study). This was done to avoid any bias or allegations of bias in our testing methods or approaches.

Q. What techniques did you use to analyze the supplements and what factors did you have to take into account? Were the results surprising?

A: All of the weight loss and men's virility products were tested using HPLC–DAD–ELSD–MSn detection with the MS in electrospray ionization (ESI)(+) and set for data dependent scanning (MS2 for weight loss and MS3 for men's virility PDE-5 inhibitors). The sports products were tested using GC–MS in electron ionization (EI) and liquid chemical ionization (CI) using methanol on an ion-trap GCMS. Ion-traps are often thought of as being desirable for tandem MS. However, we have GC triple quad systems for that. We use the ion-traps for their ability to switch between EI and CI rapidly without changing the source. That allows us to get an idea of the pseudomolecular ion as well as library searchable EI spectra. The results in this area were very surprising because some of the products containing aromatase inhibitors or anabolic steroids also contained numerous synthesis impurities or mixtures of steroidal compounds that are obviously left over from cooking the parent drug. In the future, we will probably be doing almost all of this screening on the QTOF since we can obtain both accurate mass and tandem MS data in one run.

Q. Were there limitations to your analyses? How could they be overcome?

A: We did not screen for other drug categories outside of the one that was involved in the Class I recall so there could have been other drugs in some of the samples. If we had run the samples through a general FDA screening method for HPLC connected to the QTOF, we could have run very broad searches in the multiple accurate mass databases, which would have helped to detect other potential adulterants outside the scope of those we studied. Furthermore, our generous exclusionary criteria eliminated all of the products sold on-line through Amazon and eBay stores as well as Alibaba, so there could be many more products out there that were recalled and are now being sold through those channels; we do not know how many of them are now clean and how many are tainted products.

Reference

1. P.A. Cohen et al., JAMA 312(16), 1691 (2014).

2. W. Zhang, G. Giancaspro, K.M. Adams, J. Neal-Kababick, J. Hildreth, A. Li, M.C. Roman, and J.M. Betz, J AOAC Int.97(6), 1503–13 (2014).

3. Pierre Gy, Sampling Theory and Sampling Practice, Second Edition: Heterogeneity, Sampling Correctness, and Statistical Process Control (CRC Press, 1993).

James Neal-Kababick is the founder and Director of Flora Research Laboratories (FRL), which specializes in the research and analysis of botanicals, dietary supplements and related compounds. He is also adjunct faculty at Bastyr University (USA) where he teaches botanical drug identification by microscopy and thin layer chromatography. In addition to his work at the private research lab and university, he serves on multiple expert committees such as AOAC, USP, NIH, and AHPA among others. His work is currently focused on the utilization of modern analytical technologies in the investigation of dietary supplements and other agricultural products. James is also a renowned expert in the detection of clandestine pharmaceutical adulteration of dietary supplements. He was appointed to the USP<2251> Intentional Adulteration of Dietary Supplements with Drugs and Drug Analogues Expert Committee, joining experts from the FBI, FDA, and global academia to apply the phytoforensic approach and address methodology issues for clandestine adulteration of dietary supplements.

E-mail: jimk@floraresearch.com

Website: www.floraresearch.com

This article was first published in LCGC's digital magazine, The Column. Click here to view the full issue>>